Allergic diseases, for example, asthma, rhinitis, eczema and food allergies are reaching epidemic proportions in the world. These type I hypersensitive reactions are based on the formation of immunoglobulin E (IgE) antibodies against, in principle, harmless antigens, allergens.
Immunoglobulin E molecules are produced by B cells. Each immature B cell expresses a monomeric form of IgM on its surface but all these antibodies are identical in amino acid sequence, and hence, in antigen-binding specificity. By binding to IgM antibodies the foreign multivalent antigen crosslinks B-cell (Igα/Igβ) receptors on the immature B cell surface. This triggers B-cell proliferation, differentiation into antibody-producing plasma cells, memory formation, and antigen presentation to T cells. T cells provide help for the B cells to mature which includes both isotype switching and activation of somatic mutations to improve secreted antibody affinity and selectivity. It has been observed that in allergy T helper cells differentiate to Th2 cells which produces cytokines (IL-4 and IL-13) which cause the isotype switching from IgM to IgE antibody (antibody heavy chain type is changed from μ to ε; light chain type is not changed) (Alam et al., 2003; Chaplin 2003).
The mature B cell is then able to secrete IgE antibodies which are able to bind both to antigens (allergens) but also to high affinity FcεRI receptors on a mast cell or basophil surface. The crosslinking of FcεRI complexes on the cell surface triggers the granulation of biological mediators like histamine and lipid mediators that cause inflammatory reactions (Prussin et al., 2003).
As previously described, the crosslinking of cell-surface receptors appear in two stages. Firstly, when antigen (allergen) crosslinks Igα/Igβ receptors on the immature B cell surface, and secondly, when allergen crosslinks FcεRI receptors on the mast cell surface. In addition, similar crosslinking occurs on the activation of antigen-presenting cells (APCs) like dendritic cells (Gould et al. 2008). In these cases, crosslinking is possible, when antigen (allergen) is multivalent, having multiple binding sites (epitopes) for antibodies.
The first three-dimensional immunocomplex structure of allergen and IgE antibody was recently published (Niemi et al., 2007). In this structure, dimeric β-lactoglobulin (BLG) from cow's milk (Bos d 5) is bound to two IgE/Fab fragments. Surprisingly, the IgE-binding epitope of BLG covered a flat area on the allergen surface which is unusual because according to the crystal structures the majority of known IgG epitopes are located in the protruding areas of antigens. The IgE/Fab fragments were located on the same side which makes, in principle, possible that dimeric BLG is able to link two identical IgE antibodies leading to the mast cell granulation.
This novel observation about the role of dimerization for the allergenicity of BLG lead the inventors further to investigate how common dimerization (or other type of oligomerization) is among allergens. Some allergens were reported to be oligomeric but most allergens were reported to be monomeric. It was also soon realized that there are controversial data in the literature. In corresponding cases, such as reports considering birch allergen Bet v 1, the allergen was reported to exist as monomer and sometimes as dimer. In fact, BLG is a well studied example of a transient dimer, a protein which may exist both as monomer or dimer in solution (Nooren et al., 2003). Sakurai and Goto (2002) have also studied the monomer-dimer equilibrium of BLG as a model of protein-protein interaction. Thermodynamically, the dissociation constant for the dimer is so high that in solution, BLG may exist both as monomer and dimer. Naturally, environmental factors, like pH or salt concentration affect on equilibrium. However, in physiological conditions in the body BLG is almost totally monomeric. The key element of the present invention is that transient dimerisation in many cases is a critical feature of allergens, and this transient dimerisation can be obstructed by directed mutagenesis.
Transient dimers are difficult to observe because high dissociation constant for dimer reduces the dimeric fraction to negligible at normal cellular concentrations (10-100 nM). However, by colocalization within a cell may increase concentration locally (to 1 mM), and the interaction between monomers can increase from neglible to substantial (Kuriyan et al., 2007). One example of the colocalization is the binding of antigens (allergens) on the surface of immature B cell or mast cell. This would mean that weak homodimerization of allergen on the cell's surface may be enough for signal transduction leading to sensitization or allergic reaction. The significance of allergen dimerization has previously been studied very little. Scholl et al. has studied the role of dimerization of birch pollen allergen Bet v 1 for cross-linking. Skin tests in Bet v 1-allergic mize were positive with Bet v 1 dimer, but remained negative when using the monomer. In addition, the monomer was less capable of activating murine memory B cells for IgE production in vivo. In this study, monomeric form of Bet v 1 was prepared with the addition of 4% glycerol (Scholl et al., 2005). However, the problem is that the effect of additive is only temporary because glycerol is gradually diluted and the normal monomer/dimer equilibrium is reached. Later, according to the molecular modelling analysis it was suggested that nonallergenicity of T1 protein, a member of Bet v 1 protein family was possibly based on prevented dimerization of T1 protein (Ghosh D et al., 2008). Verdino et al suggested in 2002 that the disruption of the dimer assembly of Phl p 7 allergen would diminish cross-linking. However, in the case of Phl p 7 it is in practise very difficult to create stable monomeric variant, because two polypeptide chains are very strongly associated together forming a very stable dimer (Verdino et al. 2002).
The influence of quaternary structure to allergenicity and immunogenicity of cockroach allergen Per a 3 has also been investigated. Hexameric form induced a stronger leukotrience release from basophils than the monomeric form. Unfortunately, the preparation of monomeric and hexameric forms of Per a 3 was not described in the paper (Bellinghausen et al., 2007). Another study of cockroach allergen Bla g 2 showed that dimeric wild type Bla g 2 induced more β-hexosaminidase release from mast cells than a monomeric Bla g 2 mutant (Li et al. 2008). However, the authors concluded that Bla g 2 would exist as a dimer in physiological conditions.
In de Halleux et al., 2006, it is disclosed that it is expected that a monomeric form of Der p 1 would be less prone to trigger anaphylactic reactions on injection to sensitized patients. However, experiments showing such results are not disclosed.